• Expériences d’incorporation de tritium
Les mêmes types de solution mère que lors des expériences sur le peptide H3122-135 sont utilisés, à l’exception de 3H-BPASS (2 mM et 1072 MBq.mL-1 ; AS = 536 GBq.mmol-1) ou (2 mM et 1258 MBq.mL-1 ; AS = 629 GBq.mmol-1).
Dans la glace, une solution est réalisée en ajoutant 100 µL de solution de 3H-BPASS (200 µM final), 400 µL ou 640 µL de tampon phosphate pH 7,9 10 mM, respectivement avec ou sans ajout de 140 µL de solution de H3122-135 (400 µM finale), puis 360 µL d’hAsf11-156 (200 µM finale). La solution résultante est dégazée avec N2O pendant 5 min sans buller. Le flux est coupé, et la solution est agitée doucement puis laissée à équilibrer en échangeant l’O2 dissous avec les molécules de N2O pendant 10 min. Le flux de N2O est rallumé pendant 2 min, puis recoupé. La solution est agitée doucement puis laissée à équilibrer de nouveau pendant 10 min. Cette opération est réalisée 4 fois au total. Le mélange réactionnel est irradié à 4°C avec le LINAC avec 50 impulsions (avec 3H-BPASS d’AS 536 GBq.mmol-1) ou 200 impulsions (avec 3H-BPASS d’AS= 629 GBq.mmol-1) de 20 Gy à 0,4 Hz, soit respectivement une concentration de 582 µM ou 2330 µM cumulée en HO•.
• Purification sur colonne échangeuse d’ions
La protéine irradiée est purifiée directement sur une colonne semi-préparative échangeuse d’anions Resource Q 1 mL (Amersham) selon le gradient : 0 % B en 10 min, 0 à 20 % B en 22 min et 20 à 50 % en 50 min à 1 mL.min-1 (tR Asf = 25 min) avec les éluants (A : Tampon Tris 50 mM pH 8 ; B : Tampon Tris 50 mM pH 8 / NaCl 1M), avec une détection UV à 216 et 280 nm. Les puretés UV et radiochimique sont contrôlées selon le même gradient.
• Digestion de la protéine hAsf11-156 et purification sur phase inverse C18
La protéine dans l’éluât de 2 mL est digérée avec la trypsine 1/50 (v/v) au bain-marie à 37°C pendant 10 h. Les peptides sont ensuite séparés par HPLC sur une colonne analytique (Agilent Zorbax C18, 300 Å, 250*4.6 mm) selon le gradient 2 à 50 % B en 45 min à 1 mL.min-1 avec les éluants (A : H2O / FA 0,1 % ; B : ACN / FA 0,1 %), avec une détection UV à 216 et 280 nm. Les puretés UV et radiochimique de chaque peptide sont contrôlées selon le même gradient. Les peptides sont identifiés par LC-MS. Les temps de rétention de chacun des peptides obtenus sont : 124-134 (15,0 min), 138-145, (16,0 min) 109-123 et 109-134 (21,6 min), 149-156 (22,9 min), 42-69 (31,2 min), 70-102 et 70-108 (36,2 min) et 4-41 (41,8 min).
Les peptides sélectionnés sont séquencés selon le protocole général (IV.H)
• Normalisation
Afin de comparer nos résultats, les activités spécifiques des résidus de hAsf11-156 sont normalisées selon trois facteurs correspondant au rapport entre l’activité spécifique d’un résidu donné respectivement en absence et en présence de H3122-135.
La valeur moyenne est calculée à partir des acides aminés les mieux marqués (> à 200 Bq.nmol-1) et ne présentant aucune variation d’accessibilité, soit : E124, P127, V128, K129, N152, et E154. Cette valeur moyenne est de 2.
Le plus grand rapport est observé pour le résidu E124 avec de 2,9 et le plus petit avec le résidu P127 avec 1,5 du peptide 124-ENPPVKPDFSK-134.
Les valeurs maximale (2,9), minimale (1,5) ou moyenne (2) sont appliquées (Figure III-22).
b. Essais de digestion de la protéine hAsf11-156 et de purification
Différents essais de digestion sont réalisés avec la trypsine (R, K en C-ter), l’endoprotéinase V8 (D et E en C-ter), la chymotrypsine (), le pepsine, la pronase (non spécifique) dans divers tampon en présence ou en absence de réduction et alkylation, de détergents et de dénaturants. Enzyme Quantité (m/m) T°C Tampon Durée (h) Réducteur, détergent, dénaturant. Trypsine 1/20 37 NH4HCO3 50 mM pH 7,8 2 à 24 Trypsine 1/20 37 Tris 50 mM pH 8 20
Trypsine 1/20 37 Tris 50 mM pH 8 24 R/A
Trypsine 1/20 37 Tris 50 mM pH 8 24 R/A
Urée 3M SDS 0,1 % Trypsine 1/20 37 Phosphate 50 mM pH 8 24 Trypsine 1/20 37 Phosphate 50 mM pH 8 / ACN 10 % 24 V8 1/10 à 1/50 37 Tris 50 mM pH 8 2 à 24 V8 1/20 37 Tris 50 mM pH 8 24 R/A Urée 2M V8 1/20 37 Tris 50 mM pH 8 24 R/A Urée 3M SDS 0,1 % V8 1/10 à 1/20 37 Phosphate 10 mM pH 8 24 V8 1/20 25 Phosphate 10 mM pH 8 24 V8 1/20 25 NH4HCO3 pH 7,8 24 V8 1/20 25 NH4HCO3 pH 7,8 24 Urée 3 M SDS 0,1 % V8 1/20 25 NH4HCO3 pH 7,8 24 R/A V8 1/20 25 NH4HCO3 pH 7,8 24 Urée 3 M SDS 0,1 % R/A Trypsine + V8 1/20 37 Tris 50 mM pH 8 24 Trypsine puis V8 1/20 37 Tris 50 mM pH 8 10 + 24 Chymotrypsine 1/50 37 Phosphate 10 mM pH 7,8 1, 2, 8 Chymotrypsine 1/20 25 Tris 50 mM pH 8 18, 24 Pronase 1/100 37 Tris 50 mM pH 8 2 pepsine 1/20 à 1/50 37 pH 3 (HCl) 1 et 24
L’ensemble de ces digestions est ensuite analysé par HPLC et/ou par LC-MS. En HPLC, les colonnes en phase inverse suivantes sont testées :
- Agilent Zorbax C18, 300 Å, 5 µ, 250 mm * 4,6 mm - Agilent Zorbax C8, 300 Å, 5 µ, 250 mm * 4,6 mm - Luna C18, 100 Å, 5 µ, 250 mm * 4,6 mm
- Waters Atlantis dC18, 100 Å, 5 µ, 250 mm * 4,6 mm
Différents systèmes d’élution sont utilisés à TA ou à 42°C, à un débit de 1 mL.min-1: - A: H2O/FA 0,1 %, B: ACN/FA 0,1 %
- A: H2O/TFA 0,1 %, B: ACN/TFA 0,1 % - A: Tp acetate d’ammonium pH 6, B: ACN
Certains échantillons ont aussi été analysés en LC-MS-MS ou par séquençage automatique d’Edman. La colonne HPLC utilisée en LC-MS est une colonne analytique Waters Atlantis dC18 (100 Å, 5 µ, 150 mm * 4,6 mm), avec les éluants A: H2O/FA 0,1 %, B: ACN/FA 0,1 % à 800 µ L.min-1.
Aucune des conditions testées de digestions et de purification en phase inverse n’a permis de détecter le peptide contenant V92, V94 et V96, les trois acides aminés les plus impliqués dans l’interaction avec H3122-135, à l’exception du peptide 70-102 obtenu lors de la digestion avec la trypsine. Cependant, ce peptide est trop long et est récupéré en trop faible quantité pour pouvoir l’utiliser pour analyser les résidus d’intérêt.
La synthèse du peptide AVGVTVVLITCTYRGQE contenant V94 et correspondant à un peptide théorique de la double digestion trypsine / V8 a été commandé. Ce peptide est uniquement soluble dans 100 % DMSO et peu soluble dans des systèmes H2O/ACN. Les analyses HPLC et LC-MS menées ont montré que ce peptide est très hydrophobe et reste accroché sur la colonne. Le peptide sort sur une durée de 20 min. Les tentatives de digestions et de purifications n’ont pas été menées plus loin en raison de problème de temps.
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